Hermann Strasser

Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladium and Beauveria species?

Entomopathogenic fungi are promising alternatives to chemical insecticides. However, a major hurdle concerning the registration of these fungi as plant protection agents is the possible toxicity of secreted metabolites, especially secondary metabolites. This review summarizes data on specific secondary metabolites (destruxins, efrapeptins, oosporein, beauvericin and beauveriolides) produced by the important genera Beauveria, Metarhizium and Tolypocladium . The quantities of secondary metabolites produced by these fungi in vivo are usually much less than those secreted in nutrient rich liquid media. Methods and strategies are suggested which could standardize the risk assessment of fungal biological control agents.

Monitoring the distribution of secondary metabolites produced by the entomogenous fungus Beauveria brongniartii with particular reference to oosporein

Oosporein was the only major secondary metabolite produced by three commercial isolates of the entomopathogenic fungus Beauveria brongniartii in submerged cultures and on sterilised barley kernels. None of the other toxins (bassianin, beauvericin and tenellin) normally produced by Beauveria species were detected by sensitive HPLC and MS techniques. The maximum amount of oosporein produced in batch reactors was 270 mg l−1, after 4 days incubation, while that produced on sterilised barley kernels ranged between 2.0 and 3.2 mg kg−1, after 14 days incubation. The mean amount of oosporein detected in cockchafer larvae infected with B. brongniartii was 0.23 mg. Melocont®-Pilzgerste, a commercial product based on B. brongniartii, was not phytotoxic to Lepidium sativum and Phleum pratense nor were fungal metabolites detected in these indicator plants. No systemic effects of oosporein were observed in treated pasture turf maintained for several months in the greenhouse.

A review of the natural enemies of beetles in the subtribe Diabroticina (Coleoptera: Chrysomelidae): implications for sustainable pest management

Abstract Diabroticina is a speciose subtribe of New World Chrysomelidae (Subfamily Galerucinae: Tribe Luperini) that includes pests such as corn rootworms, cucumber beetles and bean leaf beetles (e.g. Diabrotica, Acalymma, Cerotoma species). The evolution and spread of pesticide resistance, the European invasion of Diabrotica v. virgifera LeConte, and possible development of resistance due to the large-scale deployment of Diabrotica-active Bt maize in North America have generated a sense of urgency in developing biological control options against Diabroticina pests. In the present study, we review available knowledge on biological control options, including 290 publications on natural enemy–Diabroticina associations in the New World. Several natural enemy species or groups appear to be promising candidates for control strategies with different ecological rationales. We propose that future research should pursue: (1) development of inundative biological control products, particularly mass-produced entomopathogenic nematodes and fungi, (2) understanding of specific natural enemies of Diabroticina larvae throughout the Americas and of adults particularly in higher altitudes of Central America or northern South America including potential classical biological control agents against D. v. virgifera; (3) enhancement of natural enemies through cultural practices, i.e., reduced tillage, reduced weed control, cover crops, diversified crop rotations or soil amendments. Research and action must be coordinated to accelerate the exploration of biological control options.

Leaching of zinc from an industrial filter dust withPenicillium, Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

SummaryHeterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.

Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins

Nutritional conditions causing droplet exudation by Metarhizium anisopliae var. anisopliae were studied. Exudation in droplets occurred only on media with more than one carbon source and was highly dependent on the ratio of a well metabolized sugar such as trehalose and a nonpreferred sugar, in particular arabinose. Exuded droplets contained destruxin A, B and E in concentrations similar to those on submerged culture on Czapek Dox medium with equivalent C : N ratios but was clearly less than previously reported on standard Czapek Dox or Sabouraud dextrose broth. Destruxins also were found in agar samples from directly below mycelium and from up to 2 cm from the colony edge. Exudates retrieved from different media were proven to have Pr1 protease-related enzyme activity. Additional HPLC analysis indicated that droplets from diverse media did not differ in their sugar and acid content. A hypothesis is presented regarding the trigger for guttation in Metarhizium during growth under these conditions.

Leaching of iron and toxic heavy metals from anaerobically-digested sewage sludge

SummaryHeavy metal-loaded sewage sludge was leached abiotically using FeCl2 and FeCl3 which are applied in waste water treatment plants to eliminate phosphate and for coagulation. Due to the hydrolyzing nature of ferric iron, ferric chloride (100 mmll L−1) was able to solubilize more than 90% of copper and zinc and more than 80% of cadmium, with an optimal pulp density of 3% (w/v), after 10 h of exposition at 25°C. Chromium, lead and nickel were solubilized to an extent of 40–70%. With the exception of copper (redoxolysis), all heavy metals monitored were leached following the principle of acidolysis. Chemical leaching with iron resulted in a secondary contamination of sewage sludge (96 g iron per kg dry weight). The insoluble iron compounds which were precipitated for adsorbed to sludge flocks could be resolubilized with oxalic acid (100 mM, pH<3.3) up to an extent of 90%. Iron was leached by acidolysis and held in solution by complexation with oxalic acid. The pH optimum for the treatment of sewage sludge with 100 mmol L−1 oxalic acid was ≈pH 3.3. At this pH an excessive solubilization of nutrient elements and compounds (phosphorus, nitrogen, alkali and alkali earth elements) could be avoided concomitantly leaching 75% iron. Furthermore the hydrophobicity of the sewage sludge was significantly reduced as a result of treatment with iron chloride.Thiobacillus ferrooxidans (isolated from arsenopyrite and adapted on sewage sludge) utilized ferrous iron as an energy source in the presence of chloride ions (FeCl2) as efficiently as ferrous sulphate. No toxic effects of oxalic acid onT. ferrooxidans were observed at the prevailing concentration.

Proposals for Bacterial and Fungal Biocontrol Agents

Registration of biological control agents containing micro-organisms is a long-lasting and costly procedure and has discouraged companies investing in the development of microbial biological control products. Risk assessment and regulation of microbial biological plant protection products is reviewed to develop proposals for an improvement of the current system. Minimum data requirements to be presented during pre-submission meeting were defined in order to support decisions on data requirements for the dossier and possible waivers. Criteria for the possible inclusion of microbial biocontrol agents into the “low risk products” list and support for the discussion of a comparative risk analysis are discussed. Major problems are the lack of validated risk assessment methods for microbials, knowledge gaps on the natural distribution of the biocontrol micro-organisms and on natural exposure of humans and other non-target organisms, and missing definitions allowing the identification of low risk products. Potential alternative approaches for the assessment of infectivity, toxicity, identification of relevant metabolites and the risk assessment procedures for metabolites and sensitisation are discussed. Proposals for waivers for potentially obsolete data requirements related to infectivity, effect on soil biota, fate and behaviour in the environment and genetic stability are recommended.

Developing a risk indicator to comparatively assess environmental risks posed by microbial and conventional pest control agents

Abstract Selected biological control agents and conventional pesticides were used to critically review the applicability of a newly developed Risk Indicator (RI) system. Five basic components are proposed for the calculation of the overall environmental risk score: persistence of the active ingredient, dispersal potential, range of non-target organisms that are affected, and direct and indirect effects on the ecosystem. Several risk measurement systems were reviewed; risk categories in the proposed system were modified from a model developed for classical biocontrol agents. Additionally, one new category was included, to assess the risks to vertebrate non-target species. Besides a detailed discussion of the new RI model, the suitability of the model was demonstrated by calculating the risk scores for 17 selected products. It became obvious that the environmental risk score varied greatly within the assessed chemical products, and also within the group of biological products. The use pattern greatly influenced the estimated environmental risk posed by any given product. The overall environmental risk score varied between a very low risk score of 24 (Coniothyrium minitans, soil application) and a near maximum risk score of 4275 (high risk reference DDT, foliar spray). The proposed model can be used to communicate environmental risk and to design lower risk integrated pest management strategies. It is suggested that the proposed RI system may serve to define low risk and reduced risk pesticides. Yet, it remains debatable whether the RI will be useful in determining acceptability of data waivers for regulatory purposes.

Metabolite Toxicology of Fungal Biocontrol Agents

Up to 300,000 potentially unique mycotoxins were reported by the Council of Agricultural Science Technology in 2003. Nevertheless, little information is available on fungal biological control agents (FBCAs) that have been developed or are being developed. The knowledge on fungal secondary metabolites and their toxicological significance depends on what is already known in the published scientific literature or in few cases becomes apparent by chance from high throughput screening programmes during product development. The purpose of this chapter is to describe what data basis is most frequently used for a routine evaluation of fungal metabolites and their residues in FBCAs. A decision scheme is discussed, which should be used to assess metabolite toxicity in the context of a worst-case scenario testing. This risk assessment procedure will help to identify the low risk, if any, of old and newly developed microbial pest control agents, and give support to applicants to market their FBCAs.

Carbon utilization pattern as a potential quality control criterion for virulence of Beauveria brongniartii

The registered entomopathogenic fungus Beauveria brongniartii (BIPESCO 2) was tested for its virulence after one, five and 10 times sub-culturing on four types of selective synthetic nutrient media. Bioassays with third instar Melolontha melolontha larvae showed that sub-culturing negatively affects the virulence of the fungus after 10 transfers. With the Biolog SF-P2 and Biolog SF-N2 microtiter plate systems the sub-cultivated B. brongniartii conidia were monitored for any change in the carbon utilization pattern of 128 carbon sources. With the help of Spearmans rank correlation, principal components analysis and canonical correspondence analysis, respectively, six carbon sources were identified as potential virulence indicators for BIPESCO 2 (pyruvic acid, maltose, glycyl-L-glutamic acid, malonic acid, glucuronamide and phenylethylamine). The Biolog microtiter plate system is suggested as a simple and inexpensive test-system for virulence determination of B. brongniartii strain BIPESCO 2 in routine quality control.